Evolution of secondary cell number and position in the Drosophila accessory gland

In animals with internal fertilization, males transfer gametes and seminal fluid during copulation, both of which are required for successful reproduction. In Drosophila and other insects, seminal fluid is produced in the paired accessory gland (AG), the ejaculatory duct, and the ejaculatory bulb. The D. melanogaster AG has emerged as an important model system for this component of male reproductive biology. Seminal fluid proteins produced in the Drosophila AG are required for proper storage and use of sperm by the females, and are also critical for establishing and maintaining a suite of short- and long-term postcopulatory female physiological responses that promote reproductive success. The Drosophila AG is composed of two main cell types. The majority of AG cells, which are referred to as main cells, are responsible for production of many seminal fluid proteins. A minority of cells, about 4%, are referred to as secondary cells. These cells, which are restricted to the distal tip of the D. melanogaster AG, may play an especially important role in the maintenance of the long-term female post-mating response. Many studies of Drosophila AG evolution have suggested that the proteins produced in the gland evolve quickly, as does the transcriptome. Here, we investigate the evolution of secondary cell number and position in the AG in a collection of eight species spanning the entire history of the Drosophila genus. We document a heretofore underappreciated rapid evolutionary rate for both number and position of these specialized AG cells, raising several questions about the developmental, functional, and evolutionary significance of this variation.


Introduction
Along with sperm, Drosophila males transfer seminal fluid proteins (Sfps) to females.Transfer of molecules from male to female provides a fascinating example of male-female co-operation and conflict [1].Female reproduction requires seminal fluid, yet this requirement exposes females to male adaptations that may subvert their reproductive interests [2][3][4][5][6][7].Seminal fluid may also mediate competitive interactions between the sperm of multiple males in the female reproductive tract [8][9][10].Significant polymorphism of genes affecting seminal fluid function is evident in the rapid laboratory evolution of male toxicity toward females and the rapid evolution of male sperm displacement phenotypes in experimental populations of Drosophila melanogaster [2,3,11].The outcome of sperm competition is also influenced by female genetic variation and the interaction of genetic variation in the two sexes [12][13][14].Drosophila Sfps are produced in the accessory gland (AG), ejaculatory duct, and ejaculatory bulb.The molecular products of the seminal fluid producing organs exhibit rapid evolution, as expected given their role in sexual conflict [15,16] Sfp protein sequences often evolve very quickly [17], sometimes under the influence of directional selection [18][19][20][21].Expression levels of AG-biased genes may vary widely even between closely related populations and species [22][23][24][25], and the AG transcriptome exhibits rapid turnover due to gene presence/absence variation [26][27][28] or the expression of different homologues in the AG of different species [23,[29][30][31].
In most Drosophila species the AG is composed of two major cell types [32].In D. melanogaster, main cells constitute about 96% of the cells, which are the primary producers of the secreted proteins required for fertility, and induce both short-and long-term changes to female post-copulatory physiology and behavior [8,[33][34][35][36][37][38].The secondary cells (estimated to be 4% of the cells) are clustered in the distal portion of the organ (reviewed in [39]).Their secreted products appear to play an important role in maintaining female post-copulatory responses [39][40][41].Secondary cells (SC) play a role in the secretion of extracellular vesicles (i.e., exosomes), which bind to sperm, interact with the female reproductive tract epithelium, and play a role in the long-term mating response of females [42].
Recent RNA-seq analysis of secondary cells has revealed that their transcriptome is distinct from that of main cells and also evolves in a distinct manner [43,44].Given that male reproductive proteins and transcriptomes evolve quickly and that mating systems vary dramatically across Drosophila [45], whether most basic conclusions about the AG as described in the D. melanogaster model apply generally to Drosophila is an open question.A report by [32], which focused on AG main cell binucleation in multiple Drosophila species, provided incidental data (their Fig 1) strongly suggesting that Drosophila secondary cell number evolves, perhaps quickly.Given their inherent biological interest and relative ease of measurement, we set out to quantitatively investigate secondary cell number variation in a collection of Drosophila species.The phylogenetic relationships of these species is depicted in Fig 1 .Four of these species (D. melanogaster, D. simulans, D. yakuba, and D. ananassae) are from the melanogaster group; two (D.hydei and D. mojavensis) are from the repleta group; one (D.pseudoobscura) is from the obscura group, and one (D.virilis) is from the virilis group.The species were sampled to provide information about shorter and longer time scale evolutionary change.On a long timescale, the most recent common ancestor of all eight species existed about 40-50 million years ago [48,49].On a shorter timescale, divergence between D. melanogaster and D. simulans is about 2-3 million years [50].Three inbred lines of D. melanogaster were used to investigate the possibility of genetic variation affecting SC number in this species.

Dissecting, fixing, and staining glands
Virgin males were collected and then dissected two days after their estimated age of sexual maturity, which varies among species [45].D. melanogaster, D. simulans, and D. yakuba were dissected two days post-eclosion.D. hydei were dissected 10 days post eclosion.D. mojavensis, D. pseudoobscura, D. ananassae, and D. virilis were dissected eight days post eclosion.
Dissected accessory glands were fixed in 4% paraformaldehyde for 30 minutes at room temperature, and then washed with 1X PBS.The glands were treated with a 1:100 dilution of 10mg/ ml RNAse in PBT for 30 minutes at room temperature and stained using propidium iodide at a concentration of 1:1000 for 30 minutes at room temperature [32].Glands were then mounted on glass slides with spacers-Scotch tape layered and cut into squares that would mark where the corners of the 0.5mm coverslip would lie.Spacers were necessary to prevent compression of the gland.One gland of the two was imaged using a Leica confocal microscope at 20X (dry) and 40X (oil) magnification with 75-100 z-stacks per gland.The Z step size was approximately 1.8-2.0μm.Images were stitched together using the Pairwise Stitching of Images [51] or were merged at the Leica confocal upon image acquisition using the tilescan function with 10% overlap.

Secondary cell quantification
Due to the nature of the propidium iodide staining and the structure of the cell itself, SCs were easily distinguished from main cells by their characteristic large, dark vacuoles (Fig 3).SCs were counted manually across z-stack images using the Cell Counter Plugin in FIJI [52].After initializing a .tiffile in this plugin, the "show all" function allowed us to track and count each SC throughout the z plane.Two arbitrary counters, which were conveniently different colors, were used to tally the different secondary cells to properly ensure that double counting did not occur.One counter tracked one side of the epithelial layer, and after going through the lumen of the gland; the second counter was used to track cells on the other epithelial layer.The plugin then tallied the number of times the counter was used.Totals from the two counters were summed to estimate the number of secondary cells in a gland.Distinguishing between each epithelial layer was possible due to the spacers on the slide to prevent the compression of the tissue.

Accessory gland length and area
AG gland measurements were done using FIJI [52].Gland length measurements were done by using the freehand tool and drawing a line directly down the middle of the gland spanning from the top of the ejaculatory duct and the tip of the gland.The measurements were taken in pixels following [53,54].AG area was estimated by using the lasso tool on FIJI to draw perimeters around the glands, after which, measurements were made and the area in microns generated following [53,54].

Statistics and phylogeny
Statistics were done using R version 4.2.2;R Core Team 2020 /Rstudio version 2022.02.3 Build 492."Prairie Trillium" Release (1db809b8, 2022-05-20) for Windows [55].For ANOVA and Tukey's Honest Significant Difference Test, the package 'multicomp' was used [56].Violin plots were created using ggplot2 version 3.4 [57].The phylogeny was constructed using package 'ape' version 5.6.2 in R/Rstudio [58].D. pseudoobscura, our only representative of the obscura group, is sister to the melanogaster group.It exhibits fewer SC than any melanogaster subgroup species, though the difference is not significant.Much like the melanogaster subgroup, the secondary cells are enriched in the distal tip of the gland.The D. pseudoobscura accessory gland was clearly shorter and about the same width (mean length and width, 546 microns and 191 microns, respectively) compared to the melanogaster subgroup (mean length and width, 1426 microns and 189 microns, respectively).

Results and discussion
D. hydei and D. mojavensis, sister species in our sample, belong to the repleta group, a diverse clade of primarily cactophilic flies that originated about 20-30 MYA, most likely in South America [60,61].The reproductive biology of these flies differs from several other Drosophila species in multiple ways.For example, D. mojavensis and D. hydei share high re-mating rates compared to most other Drosophila species, with D. hydei apparently having a particularly high rate [45].D. hydei also has very long sperm and testis compared to most flies [62].We observed that these two species have extraordinarily high numbers of SC per gland, roughly six times as many as observed in the melanogaster group (Fig 1).Given that the D. hydei gland is only about four times the size of D. melanogaster (Table 1), it appears that the species differences in SC number cannot be explained solely by gland size variation, which is driven primarily by gland length differences.Supporting this conclusion, in striking contrast to the melanogaster group, repleta group flies exhibit SC distributed homogeneously throughout the gland (Fig 3).Interestingly, the repleta group appears to have lost the sex-peptide gene [63,64], which codes for a protein critical for proper sperm use in D. melanogaster [65].Whether the absence of sex-peptide, the increase in number of SC, and the homogeneous physical distribution of SC in the AG in the repleta group are functionally related is an interesting question.
D. virilis, which is sister to the repleta group (Fig 1 ), also exhibits substantially greater numbers of SC than the Sophophora species, though many fewer than repleta group flies (Fig 4).Thus, data from the sampled taxa are suggestive of greater numbers of SC per gland for the Drosophila group than for the Sophophora group (Fig 1), though additional sampling would be required to be confident of this inference.Similar to the repleta group species, D. virilis SC appear to be distributed homogeneously throughout the gland (Fig 3).In general, our data support the idea that restriction of the SC to the distal tip of the accessory gland in the D. melanogaster model system is a highly derived trait and not characteristic of most Drosophila lineages.The developmental basis of SC number and position, as well as their functional consequences and evolutionary processes driving divergence of these traits, are unknown.

Conclusion
While phylogenetic comparative analysis of SC number and position in the accessory gland would be necessary to make strong, specific inferences on rates of phenotypic evolution of SC number on particular branches of the Drosophila phylogeny, the results reported here leave no doubt that SC number evolves quickly in the genus.Additionally, the physical distribution of SC in the gland also evolves, as most species do not appear to share the stereotypical clustering of SC in the tip of the gland seen in the D. melanogaster model.Further investigation of the patterns presented here promise to reveal new functional and evolutionary attributes of accessory gland diversification and its broader connections to mating system biology in the genus.

Fig 1 .
Fig 1.Estimates of secondary cell number for eight Drosophila species.Phylogeny of eight Drosophila species and estimates of secondary cell number for each.Number of glands analyzed for each species are indicated next to the species name; mean and standard deviation of secondary cell number per species are shown to the right.https://doi.org/10.1371/journal.pone.0278811.g001

Fig 1
Fig 1 presents our estimates of SC number in eight species.Previous estimates of SC number in D. melanogaster were roughly 43 SC per gland[59].Our results provide a similar, though slightly greater estimate of SC number per gland, and also provide a quantitative measure of variation among genotypes.The three D. melanogaster inbred lines we assayed, which originated in Raleigh, North Carolina[46], were not significantly heterogeneous for SC number (S1 Fig).Thus, while there may be genetic variation for SC number currently segregating in D. melanogaster, we observed no evidence for this in our small sample.It is worth noting that a range of SC numbers were observed within each inbred RAL genotype (e.g., for RAL 517 the range across individuals was 29 to 56).While some of this variation could represent measurement error, developmental variation in the process leading to SC differentiation could also contribute.D. simulans, the sister species to D. melanogaster, had significantly fewer SC (Figs1 and 2).This provides strong evidence that SC numbers can evolve substantially, even on short timescales.Of course, the observation of species differences provides primae facie evidence for genetic variation affecting SC number.While our estimate of D. yakuba SC number is greater than the estimates from D. melanogaster and D. simulans, it is not significantly different from D. melanogaster.Thus, the most parsimonious explanation for the observed variation in SC number in the melanogaster subgroup is a recent decrease in SC number in D. simulans.All samples from the three melanogaster subgroup species exhibited the well documented spatial patterning previously observed in D. melanogaster, with all SC distributed in the distal tip of the gland (Fig3).